Human adenylate cyclase and use therefor

Details Human adenylate cyclase and use therefor Human adenylate cyclase and use therefor

1999-09-17

2001-03-06

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S252300, C435S252330, C435S320100, C435S369000, C536S023200

Reexamination Certificate

active

06197581

ABSTRACT:

The present invention relates to the control of cellular metabolic process by human adenylate cyclase.
Cells of multi-cellular organisms may be metabolically affected by external factors, which are usually chemical. Hormones are a well-known example of such chemical factors. Generally the external chemical factors interact with a specific receptor located on the membrane of the targeted cell. The binding event of the factor to its receptor may induce alterations in cellular metabolism via a “secondary messenger” mediator.
One of the key “secondary messengers” is cyclic AMP (cAMP) (see Sutherland, Science 177:401-407 (1972)). Cyclic AMP is produced from ATP through the action of an enzyme, adenylate cyclase. It is now known that adenylate cyclase activity may be affected by a factor/receptor binding event transmitted through an associated G protein.
Alteration of the intracellular concentration of cAMP affects many cellular reactions. For example, an increase in cAMP intracellular concentration stimulates the activity of protein kinases (enzymes that transfer terminal phosphate groups from ATP to specific sites on targeted proteins). The action of the protein kinases changes the activity or function of its substrate.
For a general review of cAMP and secondary messenger systems reference is made to “Molecular Cell Biology”, Darnell et al, 1986, Chapter 16, incorporated herein by reference.
Further investigations of cAMP as a secondary messenger revealed that an alteration in cAMP intracellular concentration was caused by the interaction of several different external factors with their distinct receptors. Further, it was found that different receptors were associated with their own particular G-protein intermediary which was itself associated with adenylate cyclase. More recent investigations have shown that there are in fact different types (isoenzymes) of adenylate cyclase, which display considerable regulatory diversity.
To date eight distinct isoenzymes of adenylate cyclase have been identified and described in the literature. The complete cDNA sequences are known for isoenzymes types 1 to 8. A review of the current understanding and knowledge of the known adenylate cyclase isoenzymes is set out in Pieroni et al, Current Opinion in Neurobiology 3:345-351 (1993); Kerwin Jr in Annual Reports in Medicinal Chemistry, Section VI, Chapter 29, Pages 287-295 (ed Venuti), (1994) and Premont, Methods in Enzymology 238:116-127 (1994).
A summary of the regulation of the known isoenzymes of adenylate cyclase is set out below in Table 1.
TABLE 1
cAMP
Isoenzyme
Regulated by
concentration
1
Ca
2+
/CaM
↑
&bgr; &ggr; dimer
↓
2
G
xi
+ PKC
↑
&bgr; &ggr; dimer
↑
PKC
↑
3
Ca
2+
/CaM
↑
4
&bgr; &ggr; dimer
↑
5
Ca
2+
↓
6
Ca
2+
↓
CaM = calmodulin
PCK = protein Kinase C
It has now been found, for the first time, that the protein phosphatase calcineurin regulates an adenylate cyclase isoenzyme.
It has further now been found that the isoenzyme regulated by calcineurin is a novel previously uncharacterised adenylate cyclase isoenzyme. The novel isoenzyme of the present invention was originally referred to as adenylate cyclase 10 (AC10), but a review of nomenclature has now caused the novel adenylate cyclase to be referred to as adenylate cyclase 9 (AC9). To avoid confusion with the different isoenzyme known before the nomenclature revision as “adenylate cyclase 9”, the novel adenylate cyclase of the present invention will herein simply be referred to as “AC”.
The nucleotide sequence encoding for mouse AC has been identified, cloned and sequenced (see Example 2). The nucleotide sequence encoding for mouse AC is given in SEQ ID No 1. The sequence is also accessible in the Genbank™ database under accession No. MMU30602 and in the EMBL database under accession No. Z50190.
A nucleotide sequence purporting to be that for human AC is described in WO-A-99/01540. The nucleotide sequence presented in WO-A-99/01540 indicates that the C-terminal portion of the protein is truncated relative to that of the mouse. We have now conducted additional work in this area and, surprisingly, have cloned and sequenced a human AC construct which does not have the truncated C-terminal portion reported in WO-A-99/01540. The sequence obtained by us for human AC is set out in SEQ ID No 98.
The present invention therefore provides a polypeptide encoded by the nucleotide sequence of SEQ ID No 98 or as set out in SEQ ID No 99 (or functional equivalents or parts of those sequences).
The term “functional equivalents” is used herein to refer to any modified version of a nucleotide or polypeptide which retains the basic function of its unmodified form. As an example, it is well-known that certain alterations in amino acid or nucleic acid sequences may not affect the polypeptide encoded by that molecule or the function of the polypeptide. It is also possible for deleted versions of a molecule to perform a particular function as well as the original molecule. Even where an alteration does affect whether and to what degree a particular function is performed, such altered molecules are included within the term “functional equivalent” provided that the function of the molecule is not so deleteriously affected as to render the molecule useless for its intended purpose.
Whilst we do not wish to be bound to theoretical considerations, it is believed that calcineurin regulates AC by removal of phosphate group(s) required for the active form of the enzyme. Thus, the adenylate cyclase activity of AC is believed to decrease in the presence of calcineurin.
In a further aspect, therefore, the present invention provides the use of calcineurin in the regulation of adenylate cyclase activity, in particular in the regulation of AC.
The activity of calcineurin is itself enhanced by the presence of Ca
2+
ions and further enhanced by the additional presence of calmodulin.
In a further aspect, the present invention provides an adenylate cyclase isoenzyme, the activity of which can be regulated by calcineurin.
Desirably the calcineurin regulatable adenylate cyclase isoenzyme is encoded by the nucleotide sequence of SEQ ID No 98, functional equivalents or parts thereof.
In a yet further aspect, the present invention provides a polynucleotide comprising a sequence derived from the sequence set out in SEQ ID No 98 or a part thereof.
The phrase “derived from” includes identical and complementary copies of the sequence of SEQ ID No 1, whether of RNA or DNA and whether in single or double-stranded form. The phrase “derived from” further includes sequences with alterations which (due to the degeneracy of the genetic code) do not affect the amino acid sequence of the polypeptide expressed, as well as sequences modified by deletions, additions or replacements of nucleotide(s) which cause no substantial deleterious affection to function (including the function of the polypeptide expressed).
The polynucleotide of the present invention includes all recombinant constructs comprising a nucleotide sequence of the invention as defined above. Such recombinant constructs may be designed to express only part of AC. The constructs may include expression control sequence(s) which differ to the control sequence(s) naturally adjoining the AC gene. Optionally, the construct may include a non-AC protein encoding region. Thus the recombinant construct includes constructs encoding for chimeric proteins, which comprise at least part of AC or a functional equivalent thereof.
In a particular embodiment, the present invention provides a vector (such as a cloning or expression vector) which comprises a recombinant construct as defined above. Vectors include conventional cloning and expression plasmids for bacterial and yeast host cells as well as virus vectors such as vaccinia, which may be useful for expression in eukaryotic cell lines. Such a vector may be used to transform a suitable host cell (either for cloning or expression purposes) and the transformed host cell also forms a further aspect of the

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